Optimization of Enzymatic Hydrolysis Process for Tobacco stem via Response Surface Methodology and its Application in Reconstituted Tobacco leaf.

To enhance lignocellulose degradation in tobacco stem fibers and improve the quality of reconstituted tobacco, this study optimized xylanase-mediated hydrolysis. Xylanase OKBK was identified as the most effective enzyme, producing a reducing sugar yield of 2.29%, significantly higher than those obtained with other tested enzymes (p< 0.05). Single-factor experiments were performed to evaluate the effects of six key parameters, including enzyme concentration, hydrolysis time, pH, solid-to-liquid ratio, temperature, and shaking speed. Plackett-Burman design identified five significant factors (p< 0.05), and the steepest ascent method was subsequently used to determine the optimal region for response surface optimization. A Box-Behnken design was then applied to develop a quadratic regression model involving temperature, hydrolysis time, and enzyme concentration, which exhibited high predictive accuracy (R²= 0.9950). The optimized conditions predicted by the model were 50 ℃, 4.5 h, and 400 U/mL, resulting in a reducing sugar yield of 21.1%, closely matching the predicted value of 21.5%. Component analysis, FTIR, and SEM characterization demonstrated that xylanase effectively disrupted the lignocellulosic structure and facilitated component degradation. Furthermore, sensory evaluation of reconstituted tobacco sheets prepared from enzymatically treated stems showed improved sensory properties, particularly reduced woody odor and smoke harshness. Overall, the optimized xylanase hydrolysis process enhanced lignocellulose degradation and sensory quality, providing a promising strategy for the high-value utilization of tobacco stem waste.